Pleiotropic functions of the transmembrane domain 6 of human melanocortin-4 receptor Hui Huang and Ya-Xiong Tao Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, 212 Greene Hall, Auburn, Alabama 36849, USA (Correspondence should be addressed to Y-X Tao; Email: taoyaxi@auburn.edu) Abstract The melanocortin-4 receptor (MC4R) is a critical regulator of energy homeostasis and has emerged as a premier target for obesity treatment. Numerous mutations in transmembrane domain 6 (TM6) of MC4R resulting in functional alterations have been identified in obese patients. Several mutagenesis studies also provided some data suggesting the importance of this domain in receptor function. To gain a better understanding of the structure–function relationship of the receptor, we performed alanine-scanning mutagenesis in TM6 to determine the functions of side chains. Of the 31 residues, two were important for cell surface expression, five were indispensable for a-melanocyte-stimulating hormone (a-MSH) and b-MSH binding, and six were important for signaling in the Gs–cAMP–PKA pathway. H264A, targeted normally to the plasma membrane, was undetectable by competitive binding assay and severely defective in basal and stimulated cAMP production and ERK1/2 phosphorylation. Nine mutants had decreased basal cAMP signaling. Seven mutants were constitutively active in cAMP signaling and their basal activities could be inhibited by two MC4R inverse agonists, Ipsen 5i and ML00253764. Five mutants were also constitutively active in the MAPK pathway with enhanced basal ERK1/2 phosphorylation. In summary, our study provided comprehensive data on the structure–function relationship of the TM6 of MC4R. We identified residues that are important for cell surface expression, ligand binding, cAMP generation, and residues for maintaining the WT receptor in active conformation. We also reported constitutive activation of the MAPK pathway and biased signaling. These data will be useful for rationally designing MC4R agonists and antagonists for treatment of eating disorders. Journal of Molecular Endocrinology (2012) 49, 237–248 Introduction The global epidemic of obesity, with a prevalence of 9 . 8% of adults worldwide (Kelly et al. 2008) and 33 . 8% of adults in the USA (Flegal et al. 2010), has become one of the most significant burdens to public health. Obesity is a major independent risk factor for cardiovascular diseases and is often associated with type 2 diabetes mellitus as well as other comorbidities (Guh et al. 2009). Though intervention of dietary control and physical activity can successfully contribute to the prevention of weight gain in some groups, it is difficult to reach long-term weight loss maintenance, especially for those carrying genetic alterations (Lemmens et al. 2008, Reinehr et al. 2009, Kraschnewski et al. 2010). Multiple genes have been identified to be associated with obesity, and mutations in the melanocortin-4 receptor (MC4R) have been characterized as the most frequent cause of monogenic obesity in humans, with a prevalence of from 0 to 6% in different ethnic backgrounds (reviewed in Tao (2010)). Therefore, the MC4R has emerged as a premier target for obesity treatment. Adopting a codominant inheritance model (O’Rahilly et al. 2003), MC4R pathogenic mutations exhibit a varied degree of penetrance, interacting with the environment (Stutzmann et al. 2008). The MC4R, which is crucial for regulating both food intake and energy expenditure (Huszar et al. 1997), is a G protein-coupled receptor (GPCR) primarily expressed in the CNS. Although the classical G protein for MC4R is the Gs, recent studies suggested that MC4R couple to all three major classes of G proteins (reviewed in Breit et al. (2011)). The Gs–cAMP–PKA and ERK1/2 signaling pathways are of particular interest because they are identified to be related to the MC4R function of energy homeostasis in vivo (Sutton et al. 2005, Czyzyk et al. 2008). Up to now, more than 150 naturally occurring mutations or common alleles of the MC4R have been identified (reviewed in Tao (2009)). They are scattered throughout the receptor, including transmembrane domain 6 (TM6). At least ten naturally occurring mutations or variants have been identified in TM6 and functionally characterized. Some of the mutants (such as L250Q, P260Q, F261S, and I269N) are retained in the endoplasmic reticulum (ER; Proneth et al. 2006, Xiang et al. 2006, Fan & Tao 2009, Tan et al. 2009, Wang & Tao 2011), and L250Q (Proneth et al. 2006, Xiang et al. 2006) and I251L (Xiang et al. 2006) are constitutively active. Most of these mutations are associated with obesity, whereas I251L confers strong protection from obesity (Stutzmann et al. 2007, 237 Journal of Molecular Endocrinology (2012) 49, 237–248 DOI: 10.1530/JME-12-0161 0952–5041/12/049–237 q 2012 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/10/2021 05:03:36PM via Massachusetts Inst of Technology